Serotonin (5-HT) Transport in Human Platelets is Modulated by Src-Catalysed Tyr-Phosphorylation of the Plasma Membrane Transporter SERT

Abstract

Background/Aim: platelets possess tightly regulated systems for serotonin (5-HT) transport. This study analysed whether the 5-HT transport mediated by the plasma-membrane transporter SERT is regulated by its Tyr-phosphorylation. Methods: 5-HT transport was determined by filtration techniques, while immunoblotting procedures were adopted for detecting the Tyr-phosphorylation of SERT in human platelet fractions. Results: 5-HT accumulation in platelets pre-treated with reserpine, which prevents the neurotransmitter transport into the dense granules, decreased upon cellular exposure to PP2 and SU6656, two structurally unrelated inhibitors of Src-kinases. By contrast, the protein Tyr-phosphatase inhibitor pervanadate increased the 5-HT accumulation. Anti-SERT immunostaining of the platelet fractions showed a major band displaying an apparent molecular mass of 50 κDa, indicating that, during the analytical procedure, SERT underwent proteolysis, which was counteracted by addition of 4 M urea in the cellular disrupting medium. The Tyr-phosphorylation degree of SERT immunoprecipitated from membrane extracts decreased by platelet treatment with SU6656 or PP2, and enhanced upon pervanadate treatment. The anti-SERT immunoprecipitates displayed anti-Src immunostaining and in vitro kinase activity towards a Src-specific peptide-substrate. Platelet treatment with PP2 or SU6656 also caused a decrease in the imipramine binding to platelets. It was concluded that the Src-mediated SERT Tyr-phosphorylation regulates the 5-HT transport by affecting the neurotransmitter binding sites.

Tyrosine Phosphorylation of the Human Serotonin Transporter: A Role in the Transporter Stability and Function

“…Indeed, our phosphorylation studies established that SERT proteins are phosphorylated on tyrosine(s) in platelets and in HTR heterologous cell preparations. In agreement with our studies, Zarpellon et al (2008) provided evidence that Srcfamily tyrosine kinases regulate SERT function and SERT phosphorylation on tyrosine residues. Therefore, it is possible that regulating Src-mediated tyrosine phosphorylation of SERT or protecting from dephosphorylation by sustaining the tyrosine phosphorylated form of SERT might affect SERT protein stability and hence the function.…”

“…In addition to the role Ser/Thr protein kinases play in the regulation of SERT, evidence suggests that a family of tyrosine protein kinases also regulate SERT activity. Several structurally distinct tyrosine kinase inhibitors, such as genistein, herbimycin A, and 2,5-dihydroxycinnamate, inhibit 5-HT uptake in platelets (Helmeste and Tang, 1995;Zarpellon et al, 2008). However, the exact sequence of molecular events that occurs during the SERT regulatory process and whether SERT tyrosine phosphorylation regulates SERT activity are unknown.…”

“…It is also possible that Src regulates SERT phosphorylation and functions through direct binding to SERT. Zarpellon et al (2008) have identified the presence of Src-kinase in SERT immunoprecipitates from human platelets suggesting that SERT and Src exist as a complex.…”

“…Previous studies using human platelets indicated that although acute tyrosine kinase inhibition decreased 5-HT uptake, inhibition of tyrosine phosphatase increased uptake (Helmeste and Tang, 1995;Zarpellon et al, 2008). Because persistent activation or inhibition of cellular signaling cascades can differentially affect SERT regulation we initially examined the effects of short and prolonged (30 min versus 24 h) inhibition of protein tyrosine kinase(s) and protein tyrosine phosphatase(s) on SERT activity and expression using rat platelet-rich plasma.…”

“…5-HT neurons express Src-family kinases as well as tyrosine kinase receptors (Madhav et al, 2001) raising the possibility that stimulation of presynaptic receptors located on 5-HT neurons may regulate SERT via tyrosine kinasemediated pathways. Although short-and long-term treatment with tyrosine kinase inhibitors or activators regulate SERT function and gene transcription (Helmeste and Tang, 1995;Kekuda et al, 1997;Prasad et al, 1997;Zarpellon et al, 2008), the precise molecular basis underlying tyrosine kinase-mediated SERT regulation is not clear. The SERT sequence contains four tyrosine residues (Tyr47, Tyr142, Tyr350, and Tyr358) that face the inside of the cell and could be potential phosphorylation targets for tyrosine kinase(s) (Ramamoorthy et al, 1993a).…”